This research project will test the hypothesis that, in the diabetic milieu, S100A9 induces the calcification potential of valvular interstitial cell (VIC)-derived matrix vesicles, precursors of microcalcification, offering the novel mechanism of calcific aortic valve disease (CAVD). Our pilot studies showed that S100A9 - a recently identified biomarker of acute cardiovascular events - is expressed by VIC and a component of matrix vesicles. The present study will examine in vivo and in vitro the role of interplay between S100A9 and matrix vesicles in aortic valve calcification in diabetes. A lack of high-resolution imaging techniques that can detect preclinical microcalcification is a critical barrier to the treatment of CAVD. Our published studie linked inflammation and calcification and showed that molecular imaging and micro-optical coherence tomography (OCT) can identify microcalcification undetectable by other imaging modalities. Specific Aim 1 will test the hypothesis in vivo that S100A9 promotes the formation of microcalcification in aortic valves of diabetic mice. We expect that genetic deletion of S100A9 will reduce diabetes-triggered matrix vesicle release, microcalcification, and CAVD. Specific Aim 2 will develop novel imaging to evaluate quantitatively the impact of microcalcification on CAVD in diabetic mice and humans. Specific Aim 3 will examine in vitro S100A9-mediated mechanisms for the formation of calcifying matrix vesicles. These complementary studies will advance the field by identifying the role of S100A9 in microcalcification. The findings from this project will bolster support for S100A9 as a therapeutic target for CAVD. In addition, this project will provide bases for the development of imaging for early diagnosis of CAVD in large animals and human patients. (End of Abstract)